1. Field of the Invention
The present invention relates to murine α(1,3)fucosyltransferases, Fuc-TVII, DNA encoding such a murine α(1,3)fucosyltransferase Fuc-TVII, plasmids containing such DNA, cells transformed with such a plasmid, a method for producing a murine α(1,3)fucosyltransferase Fuc-TVII by culturing such cells, monoclonal antibodies which specifically bind to a murine α(1,3)fucosyltransferase Fuc-TVII, and immunoassays for detecting a murine α(1,3)fucosyltransferase Fuc-TVII using such monoclonal antibodies.
2. Discussion of the Background
Cell adhesion events between leukocytes and endothelial cells operate to facilitate the exit of blood leukocytes from the vascular tree. The selectin family of cell adhesion molecules, and their counter-receptors, function early in this process, mediating transient adhesive contacts between leukocytes and the endothelial cell monolayer. These selectin-dependent adhesive contacts, together with shear forces impinging upon the leukocyte, cause the leukocyte to “roll” along the endothelial monolayer. Leukocyte rolling, in turn, facilitates subsequent events that include leukocyte activation, firm leukocyte-endothelial cell attachment, and transendothelial migration (T. A. Springer, Cell, vol. 76, pp. 301-314 (1994); R. P. McEver et al, J. Biol. Chem., vol. 270, pp. 11025-11028 (1995)).
E- and P-selectin, expressed by activated vascular endothelial cells, recognize glycoprotein counter-receptors displayed by leukocytes. Each of these selectins can operate to mediate leukocyte rolling in the context of inflammation. L-selectin has also been implicated in mediating leukocyte adhesion to activated vascular endothelium, through interactions with an as yet poorly understood endothelial cell ligand (M. L. Arbones et al, Immunity, vol. 1, pp. 247-260 (1994); K. Ley et al, J. Exp. Med., vol. 181, pp. 669-675 (1995)). By contrast, lymphocyte L-selectin recognizes glycoprotein counter-receptors displayed by specialized cuboidal endothelial cells that line high endothelial venules (HEV) within lymph nodes and Peyer's patches. L-selectin-dependent adhesive interactions in this context operate to facilitate trafficking of lymphocytes (lymphocyte “homing”) to such lymphoid aggregates.
The NH2-terminal C-type mammalian lectin domain common to each of the three selectin family members mediates cell adhesion through calcium dependent interactions with specific oligosaccharide ligands, displayed by leukocytes (E- and P-selectin ligands) (R. P. McEver et al, J. Biol. Chem., vol. 270, pp. 11025-11028 (1995); A. Varki, Proc. Natl. Acad. Sci. U.S.A., vol. 91, pp. 7390-7397 (1994)), or by HEV (L-selectin) (S. D. Rosen et al, Curr. Opin. Cell Biol., vol. 6, pp. 663-673 (1994)). Physiological ligand activity for E- and P-selectins is critically dependent on the expression of a non-reducing terminal tetrasaccharide termed sialyl Lewis x (sLex) [NeuNAcα2,3Galβ1,4(Fucα1,3)GlcNAc-R] (A. Varki, Proc. Natl. Acad. Sci. U.S.A., vol. 91, pp. 7390-7397 (1994)), and/or its difucosylated variant (T. P. Patel et al, Biochemistry, vol. 33, pp. 14815-14824 (1994)). However, E- and P-selectins recognize this oligosaccharide in different contexts. P-selectin-dependent cell adhesion is optimal when sLex is displayed by serine and threonine-linked oligosaccharides residing on a specific protein termed P-selectin glycoprotein ligand 1(PSGL-1) (K. L. Moore et al, J. Cell. Biol., vol. 188, pp. 445-456 (1992); D. Sako et al, Cell, vol. 75, pp. 1179-1186 (1993)). sLex-modified PSGL-1 also appears to represent a high affinity counter-receptor for E-selectin (D. Asa et al, J. Biol. Chem., vol. 270, pp. 11662-11670 (1995); K. D. Patel et al, J. Clin. Invest., vol. 96, pp. 1887-1896 (1995)). A distinct leukocyte glycoprotein termed E-selectin ligand 1 (ESL-1) (M. Steegmaler et al, Nature, vol. 373, pp. 615-620 (1995)), and its α(1,3)fucosylated, asparagine-linked oligosaccharides, may also function as an E-selectin counter-receptor.
Physiological L-selectin counter-receptors on HEV are represented by the glycoproteins, GlyCAM-1 (L. A. Lasky et al, Cell, vol. 69, pp. 927-938 (1992)), CD34 (S. Baumhueter et al, Science, vol. 262, pp. 436-438 (1993)), and MadCAM-1 (E. L. Berg et al, Nature, vol. 366, pp. 695-698 (1993)). Biochemical studies indicate that L-selectin ligand activity of these molecules is also critically dependent upon post-translational modification by glycosylation. Early studies documented a requirement for sialylation and sulfation (Y. Imai et al, J. Cell Biol., vol. 113, pp. 1213-1221 (1991)), implied a requirement for α(1,3)fucosylation, and indicated that these modifications are components of serine and/or threonine-linked glycans. More recent oligosaccharide structural analyses extend this work, and imply that high affinity L-selectin ligand activity depends upon the sulfated variant of the sLex determinant, NeuNAcα2,3(SO46)Galβ1,4(Fucα1,3)GlcNAc-R (S. Hemmerich et al, Biochemistry, vol. 33, pp. 4820-4829 (1994); S. Hemmerich et al, Biochemistry, vol. 33, pp. 4830-4835 (1994); S. Hemmerich et al, J. Biol. Chem., vol. 270, pp. 12035-12047 (1995)).
Expression of sLex is determined by cell lineage-specific expression of one or more α(1,3)fucosyltransferases (S. Natsuka et al, Curr. Opin. Struc. Biol., vol. 4, pp. 683-691 (1994)). These enzymes utilize the donor substrate GDP-fucose, and catalyze a transglycosylation reaction involving the addition of α1,3-linked fucose to a common 3′-sialyl-N-acetyl-lactosamine precursor. It can be presumed that expression of the sulfated variant of sLex also depends upon lineage-specific expression of α(1,3)fucosyltransferase activities operating on sulfate-modified 3′-sialyl-N-acetyl-lactosamine precursors, or that create sLex moieties modified subsequently by sulfation.
The identity of the α(1,3)fucosyltransferase(s) responsible for selectin ligand expression in leukocytes is not well-defined, and HEV-specific α(1,3)fucosyltransferases have not been described. To date, five different human α(1,3)fucosyltransferases have been cloned (J. F. Kukowska-Latallo et al, Genes & Dev., vol. 4, pp. 1288-1303 (1990); B. W. Weston et al, J. Biol. Chem., vol. 267, pp. 24575-24584 (1992); B. W. Weston et al., J. Biol. Chem., vol. 267, pp. 4152-4160 (1992); J. B. Lowe et al, J. Biol. Chem., vol. 266, pp. 17467-17477 (1991); S. E. Goelz et al, Cell, vol. 63, pp. 1349-1356 (1990); R. Kumar et al, J. Biol. Chem., vol. 266, pp. 21777-21783 (1991); S. Natsuka et al, J. Biol. Chem., vol. 269, pp. 16789-16794 (1994); K. Sasaki et al, J. Biol. Chem., vol. 269, pp. 14730-14737 (1994)). Northern blot and molecular cloning analyses imply that two of these, termed Fuc-TIV (J. B. Lowe et al, J. Biol. Chem., vol. 266, pp. 17467-17477 (1991); S. E. Goelz et al, Cell, vol. 63, pp. 1349-1356 (1990); R. Kumar et al, J. Biol. Chem., vol. 266, pp. 21777-21783 (1991)) and Fuc-TVII (S. Natsuka et al, J. Biol. Chem., vol. 269, pp. 16789-16794 (1994); K. Sasaki et al, J. Biol. Chem., vol. 269, pp. 14730-14737 (1994)), are expressed in leukocyte cells, and represent candidates for critical participation in selectin ligand expression. The role of Fuc-TIV (also known as ELAM-1 Ligand Fucosyl Transferase, or ELFT) in this process is not clear, however. While Fuc-TIV/ELFT is able to efficiently utilize non-sialylated N-acetyl-lactosamine precursors to direct expression of the Lex moiety (J. B. Lowe et al, J. Biol. Chem., vol. 266, pp. 17467-17477 (1991); R. Kumar et al, J. Biol. Chem., vol. 266, pp. 21777-21783 (1991)), this enzyme cannot determine Lex expression in all cellular contexts (S. Goelz et al, J. Biol. Chem., vol. 269, pp. 1033-1040 (1994)), and its ability to do so in leukocytes, or in leukocyte progenitors, has not been demonstrated. By contrast, Fuc-TVII is apparently able to determine sLex expression in all mammalian cellular contexts examined, where sLex synthesis is biochemically possible (S. Natsuka et al, J. Biol. Chem., vol. 269, pp. 16789-16794 (1994); K. Sasaki et al, J. Biol. Chem., vol. 269, pp. 14730-14737 (1994)). Neither enzyme has been tested for its ability to participate in the synthesis of L-selectin ligands represented by the sulfated sLex determinant.
Thus, there remains a need for additional α(1,3)fucosyltransferases and methods, cells, plasmids, and DNA useful for preparing the same. There also remains a need for antibodies and immunoassays useful for detecting such α(1,3)fucosyltransferases.